Dephosphorylation of the pre-initiation complex is critical for origin firing.

In eukaryotes, cyclin-dependent kinase (CDK) ensures that the genome is duplicated exactly once by inhibiting helicase loading factors before activating origin firing. CDK activates origin firing by phosphorylating two substrates, Sld2 and Sld3, forming a transient and limiting intermediate-the pre-initiation complex (pre-IC). Here, we show in the budding yeast Saccharomyces cerevisiae that the CDK phosphorylations of Sld3 and Sld2 are rapidly turned over during S phase by the PP2A and PP4 phosphatases. PP2ARts1 targets Sld3 specifically through an Rts1-interaction motif, and this targeted dephosphorylation is important for origin firing genome-wide, for formation of the pre-IC at origins and for ensuring that Sld3 is dephosphorylated in G1 phase. PP2ARts1 promotes replication in vitro, and we show that targeted Sld3 dephosphorylation is critical for viability. Together, these studies demonstrate that phosphatases enforce the correct ordering of replication factor phosphorylation and in addition to kinases are also key drivers of replication initiation.

Selective social interactions and speed-induced leadership in schooling fish.

Animals moving together in groups are believed to interact among each other with effective social forces, such as attraction, repulsion, and alignment. Such forces can be inferred using "force maps," i.e., by analyzing the dependency of the acceleration of a focal individual on relevant variables. Here, we introduce a force map technique suitable for the analysis of the alignment forces experienced by individuals. After validating it using an agent-based model, we apply the force map to experimental data of schooling fish. We observe signatures of an effective alignment force with faster neighbors and an unexpected antialignment with slower neighbors. Instead of an explicit antialignment behavior, we suggest that the observed pattern is the result of a selective attention mechanism, where fish pay less attention to slower neighbors. This mechanism implies the existence of temporal leadership interactions based on relative speeds between neighbors. We present support for this hypothesis both from agent-based modeling as well as from exploring leader-follower relationships in the experimental data.

Checkpoint inhibition of origin firing prevents DNA topological stress.

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

COVID-19-related hyperglycemia is associated with infection of hepatocytes and stimulation of gluconeogenesis.

Occurrence of hyperglycemia upon infection is associated with worse clinical outcome in COVID-19 patients. However, it is still unknown whether SARS-CoV-2 directly triggers hyperglycemia. Herein, we interrogated whether and how SARS-CoV-2 causes hyperglycemia by infecting hepatocytes and increasing glucose production. We performed a retrospective cohort study including patients that were admitted at a hospital with suspicion of COVID-19. Clinical and laboratory data were collected from the chart records and daily blood glucose values were analyzed to test the hypothesis on whether COVID-19 was independently associated with hyperglycemia. Blood glucose was collected from a subgroup of nondiabetic patients to assess pancreatic hormones. Postmortem liver biopsies were collected to assess the presence of SARS-CoV-2 and its transporters in hepatocytes. In human hepatocytes, we studied the mechanistic bases of SARS-CoV-2 entrance and its gluconeogenic effect. SARS-CoV-2 infection was independently associated with hyperglycemia, regardless of diabetic history and beta cell function. We detected replicating viruses in human hepatocytes from postmortem liver biopsies and in primary hepatocytes. We found that SARS-CoV-2 variants infected human hepatocytes in vitro with different susceptibility. SARS-CoV-2 infection in hepatocytes yields the release of new infectious viral particles, though not causing cell damage. We showed that infected hepatocytes increase glucose production and this is associated with induction of PEPCK activity. Furthermore, our results demonstrate that SARS-CoV-2 entry in hepatocytes occurs partially through ACE2- and GRP78-dependent mechanisms. SARS-CoV-2 infects and replicates in hepatocytes and exerts a PEPCK-dependent gluconeogenic effect in these cells that potentially is a key cause of hyperglycemia in infected patients.

Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial immunometabolic modulation.

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs' intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.

Harder, better, faster, stronger: biochemistry and biophysics in the immunosurveillance concert.

Antitumor immunosurveillance is triggered by immune cell recognition of characteristic biochemical signals on the surfaces of cancer cells. Recent data suggest that the mechanical properties of cancer cells influence the strength of these signals, with physically harder target cells (more rigid) eliciting better, faster, and stronger cytotoxic responses against metastasis. Using analogies to a certain electronic music duo, we argue that the biophysical properties of cancer cells and their environment can adjust the volume and tone of the antitumor immune response. We also consider the potential influence of biomechanics-based immunosurveillance in disease progression and posit that targeting the biophysical properties of cancer cells in concert with their biochemical features could increase the efficacy of immunotherapy.

Viral-based animal models in polyglutamine disorders.

Polyglutamine disorders are a complex group of incurable neurodegenerative disorders caused by an abnormal expansion in the trinucleotide cytosine-adenine-guanine tract of the affected gene. To better understand these disorders, our dependence on animal models persists, primarily relying on transgenic models. In an effort to complement and deepen our knowledge, researchers have also developed animal models of polyglutamine disorders employing viral vectors. Viral vectors have been extensively used to deliver genes to the brain, not only for therapeutic purposes but also for the development of animal models, given their remarkable flexibility. In a time- and cost-effective manner, it is possible to use different transgenes, at varying doses, in diverse targeted tissues, at different ages, and in different species, to recreate polyglutamine pathology. This paper aims to showcase the utility of viral vectors in disease modelling, share essential considerations for developing animal models with viral vectors, and provide a comprehensive review of existing viral-based animal models for polyglutamine disorders.

Chronic psychosocial stress triggers microglial-/macrophage-induced inflammatory responses leading to neuronal dysfunction and depressive-related behavior.

Chronic environmental stress and traumatic social experiences induce maladaptive behavioral changes and is a risk factor for major depressive disorder (MDD) and various anxiety-related psychiatric disorders. Clinical studies and animal models of chronic stress have reported that symptom severity is correlated with innate immune responses and upregulation of neuroinflammatory cytokine signaling in brain areas implicated in mood regulation (mPFC; medial Prefrontal Cortex). Despite increasing evidence implicating impairments of neuroplasticity and synaptic signaling deficits into the pathophysiology of stress-related mental disorders, how microglia may modulate neuronal homeostasis in response to chronic stress has not been defined. Here, using the repeated social defeat stress (RSDS) mouse model we demonstrate that microglial-induced inflammatory responses are regulating neuronal plasticity associated with psychosocial stress. Specifically, we show that chronic stress induces a rapid activation and proliferation of microglia as well as macrophage infiltration in the mPFC, and these processes are spatially related to neuronal activation. Moreover, we report a significant association of microglial inflammatory responses with susceptibility or resilience to chronic stress. In addition, we find that exposure to chronic stress exacerbates phagocytosis of synaptic elements and deficits in neuronal plasticity. Importantly, by utilizing two different CSF1R inhibitors (the brain penetrant PLX5622 and the non-penetrant PLX73086) we highlight a crucial role for microglia (and secondarily macrophages) in catalyzing the pathological manifestations linked to psychosocial stress in the mPFC and the resulting behavioral deficits usually associated with depression.

Validation and incorporation of digital entheses into a preliminary GLobal OMERACT Ultrasound DActylitis Score (GLOUDAS) in psoriatic arthritis.

OBJECTIVES: The main objective was to generate a GLobal OMERACT Ultrasound DActylitis Score (GLOUDAS) in psoriatic arthritis and to test its reliability. To this end, we assessed the validity, feasibility and applicability of ultrasound assessment of finger entheses to incorporate them into the scoring system. METHODS: The study consisted of a stepwise process. First, in cadaveric specimens, we identified enthesis sites of the fingers by ultrasound and gross anatomy, and then verified presence of entheseal tissue in histological samples. We then selected the entheses to be incorporated into a dactylitis scoring system through a Delphi consensus process among international experts. Next, we established and defined the ultrasound components of dactylitis and their scoring systems using Delphi methodology. Finally, we tested the interobserver and intraobserver reliability of the consensus- based scoring systemin patients with psoriatic dactylitis. RESULTS: 32 entheses were identified in cadaveric fingers. The presence of entheseal tissues was confirmed in all cadaveric samples. Of these, following the consensus process, 12 entheses were selected for inclusion in GLOUDAS. Ultrasound components of GLOUDAS agreed on through the Delphi process were synovitis, tenosynovitis, enthesitis, subcutaneous tissue inflammation and periextensor tendon inflammation. The scoring system for each component was also agreed on. Interobserver reliability was fair to good (κ 0.39-0.71) and intraobserver reliability good to excellent (κ 0.80-0.88) for dactylitis components. Interobserver and intraobserver agreement for the total B-mode and Doppler mode scores (sum of the scores of the individual abnormalities) were excellent (interobserver intraclass correlation coefficient (ICC) 0.98 for B-mode and 0.99 for Doppler mode; intraobserver ICC 0.98 for both modes). CONCLUSIONS: We have produced a consensus-driven ultrasound dactylitis scoring system that has shown acceptable interobserver reliability and excellent intraobserver reliability. Through anatomical knowledge, small entheses of the fingers were identified and histologically validated.

Dual action of benzaldehydes: Inhibiting quorum sensing and enhancing antibiotic efficacy for controlling Pseudomonas aeruginosa biofilms.

Quorum sensing (QS) has a central role in biofilm lifestyle and antimicrobial resistance, and disrupting these signaling pathways is a promising strategy to control bacterial pathogenicity and virulence. In this study, the efficacy of three structurally related benzaldehydes (4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde)) in disrupting the las and pqs systems of Pseudomonas aeruginosa was investigated using bioreporter strains and computational simulations. Additionally, these benzaldehydes were combined with tobramycin and ciprofloxacin antibiotics to evaluate their ability to increase antibiotic efficacy in preventing and eradicating P. aeruginosa biofilms. To this end, the total biomass, metabolic activity and culturability of the biofilm cells were determined. In vitro assays results indicated that the aromatic aldehydes have potential to inhibit the las and pqs systems by > 80 %. Molecular docking studies supported these findings, revealing the aldehydes binding in the same pocket as the natural ligands or receptor proteins (LasR, PQSA, PQSE, PQSR). Benzaldehydes were shown to act as virulence factor attenuators, with vanillin achieving a 48 % reduction in pyocyanin production. The benzaldehyde-tobramycin combination led not only to a 60 % reduction in biomass production but also to a 90 % reduction in the metabolic activity of established biofilms. A similar result was observed when benzaldehydes were combined with ciprofloxacin. 4-Hydroxybenzaldehyde demonstrated relevant action in increasing biofilm susceptibility to ciprofloxacin, resulting in a 65 % reduction in biomass. This study discloses, for the first time, that the benzaldehydes studied are potent QS inhibitors and also enhancers of antibiotics antibiofilm activity against P. aeruginosa.

Loss of the abasic site sensor HMCES is synthetic lethal with the activity of the APOBEC3A cytosine deaminase in cancer cells.

Analysis of cancer mutagenic signatures provides information about the origin of mutations and can inform the use of clinical therapies, including immunotherapy. In particular, APOBEC3A (A3A) has emerged as a major driver of mutagenesis in cancer cells, and its expression results in DNA damage and susceptibility to treatment with inhibitors of the ATR and CHK1 checkpoint kinases. Here, we report the implementation of CRISPR/Cas-9 genetic screening to identify susceptibilities of multiple A3A-expressing lung adenocarcinoma (LUAD) cell lines. We identify HMCES, a protein recently linked to the protection of abasic sites, as a central protein for the tolerance of A3A expression. HMCES depletion results in synthetic lethality with A3A expression preferentially in a TP53-mutant background. Analysis of previous screening data reveals a strong association between A3A mutational signatures and sensitivity to HMCES loss and indicates that HMCES is specialized in protecting against a narrow spectrum of DNA damaging agents in addition to A3A. We experimentally show that both HMCES disruption and A3A expression increase susceptibility of cancer cells to ionizing radiation (IR), oxidative stress, and ATR inhibition, strategies that are often applied in tumor therapies. Overall, our results suggest that HMCES is an attractive target for selective treatment of A3A-expressing tumors.

Montelukast and cefoperazone act as antiquorum sensing and antibiofilm agents against Pseudomonas aeruginosa.

AIMS: Drug repurposing is an attractive strategy to control biofilm-related infectious diseases. In this study, two drugs (montelukast and cefoperazone) with well-established therapeutic applications were tested on Pseudomonas aeruginosa quorum sensing (QS) inhibition and biofilm control. METHODS AND RESULTS: The activity of montelukast and cefoperazone was evaluated for Pqs signal inhibition, pyocyanin synthesis, and prevention and eradication of Ps. aeruginosa biofilms. Cefoperazone inhibited the Pqs system by hindering the production of the autoinducer molecules 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal or PQS), corroborating in silico results. Pseudomonas aeruginosa pyocyanin production was reduced by 50%. The combination of the antibiotics cefoperazone and ciprofloxacin was synergistic for Ps. aeruginosa biofilm control. On the other hand, montelukast had no relevant effects on the inhibition of the Pqs system and against Ps. aeruginosa biofilm. CONCLUSION: This study provides for the first time strong evidence that cefoperazone interacts with the Pqs system, hindering the formation of the autoinducer molecules HHQ and PQS, reducing Ps. aeruginosa pathogenicity and virulence. Cefoperazone demonstrated a potential to be used in combination with less effective antibiotics (e.g. ciprofloxacin) to potentiate the biofilm control action.

POC device for rapid oral pH determination based on a smartphone platform.

Salivary pH serves as a valuable and useful diagnostic marker for periodontal disease, as it not only plays a critical role in disease prevention but also in its development. Typically, saliva sampling is collected by draining and spitting it into collection tubes or using swabs. In this study, we have developed a Point-of-Care (POC) device for in situ determination of oral pH without the need for complex instruments, relying solely on a smartphone as the detection device. Our system utilizes a non-toxic vegetable colourimetric indicator, immobilized on a chitosan membrane located on a disposable stick, enabling direct sampling within the buccal cavity. An ad hoc designed 3D-printed attachment is used to ensure accurate positioning and alignment of the stick, as well as isolation from external lighting conditions. A custom-developed smartphone application captures and automatically processes the image of the sensing membrane, providing the salivary pH results. After optimizing the cocktail composition, the developed sensors demonstrated the capacity to determine pH within a range of 5.4 to 8.1 with a remarkable precision of 0.6%, achieving a very short analysis time of just 1 min. A stability study conducted on the sensing membranes revealed a lifetime of 50 days. To validate the performance of our analytical device, we compared its results against those obtained from a calibrated pH-meter, using a group of individuals. The device exhibited an average error of 2.4% when compared with the pH-meter results, confirming its reliability and accuracy.

The meaning in loss protocol: A clinical trial of online grief therapy.

For a minority of the bereaved, the loss of a significant other can trigger an overwhelming emotional reaction and impaired functioning across life domains, known as prolonged grief disorder (PGD). Hence, ongoing efforts have been made to refine existing treatments to increase their efficacy and to accommodate the idiosyncrasies of grief reactions. This study presents the results of an open clinical trial of the feasibility and effectiveness of the Meaning in Loss (MIL) protocol in an online format. The brief intervention of 12 to 16 sessions combines constructivist and narrative strategies to explore and work through impediments to meaning reconstruction in loss. The sample included 25 participants diagnosed with PGD who were treated by six therapists. Baseline and post-therapy comparisons showed a significant improvement in all clinical measures (grief symptomatology, depression and general distress) and an increase of meaning making regarding the loss. Meaning making was found to be a prospective mediator of symptomatic improvement in grief across the course of therapy. These findings suggest the effectiveness of the MIL protocol in decreasing grief specific and associated symptomatology and argue for the relevance of further controlled evaluations of its efficacy. Moreover, results confirm previous findings that meaning making is a relevant factor in the evolution of grief reactions, including in the context of psychotherapy.

New hard tick (Acari: Ixodidae) reports and detection of Rickettsia in ticks from Sierra Nevada de Santa Marta, Colombia.

The Sierra Nevada de Santa Marta (SNSM), located in northern Colombia, is considered a geographical island with high levels of biodiversity and endemism. However, little is known about tick species and their associated microorganisms at the SNSM. In this study we sampled host-seeking ticks in areas of the town of Minca within the SNSM. We collected 47 ticks identified as Amblyomma pacae, Amblyomma longirostre, Amblyomma ovale, Amblyomma mixtum, Haemaphysalis juxtakochi, Ixodes sp. cf. Ixodes affinis and Ixodes sp. Of these ticks, we tested for Rickettsia spp. by amplifying the gltA, SCA1, and 16S rRNA genes via PCR. Rickettsia amblyommatis was detected in one pool of 3 larvae and in a female of A. pacae. Additonally, we isolated Rickettsia sp. belonging to the group of spotted fevers in larvae of A. longirostre. This study reports new findings of six species of ticks and two species of Rickettsia within the SNSM.

Physiological synchronization and innovative moments in psychotherapy: A single-case study of micro-process.

OBJECTIVE: Interpersonal synchronization is increasingly studied as a biomarker of empathy, therapeutic alliance, and treatment outcome. However, most studies average data over sessions, leaving associations between synchrony and actual interactions largely unexplored. We aim to showcase a novel approach examining synchronization during specific micro-processes: Innovative Moments (IM) as markers of exceptions to clients' problematic patterns of meaning. METHODS: Electrodermal activity was recorded over 15 sessions of a psychodynamic psychotherapy single case. Moment-to-moment patient-therapist synchrony was calculated using the Adaptive Matching Interpolated Correlations (AMICo) algorithm. The Innovative Moments Coding System was utilized to identify IMs within session transcripts with precise timing. Monte-Carlo permutation tests were conducted to examine the association between physiological synchrony and IM Levels of increasing complexity (Levels 1-3). RESULTS: Higher-than-random synchronization emerged during Level 3 IMs (p = 0.046; d = 0.21) but not in lower Levels. Post-hoc qualitative analyses linked high synchrony to sub-processes of Level 3 IMs, such as positive contrasts and attributions for change. CONCLUSION: Our findings show it is possible to link moment-by-moment physiological co-regulation to theoretically identified meaning-making processes. While generalization of these observations is undue, this work demonstrates a robust and promising application of a multimodal approach to investigating psychotherapy, providing insights into both the clinical case and the theoretical model adopted.

Client resources and the prediction of therapeutic change using an interview: An exploratory study.

OBJECTIVES: Although psychotherapy research suggests that clients' resources are related to positive outcomes, there is a lack of clinical tools available to consider their integration into psychotherapy. In this exploratory research, we studied the feasibility of a semi-structured interview to identify resources reported by clients at the onset of therapy and the relationship between resources and therapy outcomes. METHODS: Data consisted of interviews with 30 clients from a clinical trial, in which elicitation of resources and their relationship with the outcomes were the main study objectives. RESULTS: This interview was content analyzed and both adaptative resources and maladaptive resources (dysfunctional coping strategies) were identified. The association between the adaptive resources and the evolution of outcomes throughout treatment was analyzed. Time (i.e. sessions) and resources were negatively correlated with psychological distress. Moreover, resources positively influenced the impact of time on distress. CONCLUSIONS: Clinicians should not take at face value resources that are self-reported, as they may reflect the maladaptive functioning of the client. The finding that clients with higher resources at onset have better outcomes points to the need to study how resources may be elicited effectively during therapy, and if this improves psychotherapy outcomes.

Limited vs. Extended Resection of Stanford Type A Acute Aortic Dissections.

Background and Objectives: This is a propensity-matched, single-center study of limited versus extended resection for type A acute aortic dissection (AAAD). Materials and Methods: This study collected retrospective data for 440 patients with acute type A aortic dissection repairs (limited resection, LR-215; extended resection, ER-225), of which 109 pairs were propensity-matched to LR versus ER. Multivariate analysis was performed for inpatient death, long-term survival and the composite outcome of inpatient death/TIA/stroke. Kaplan-Meier survival curves were compared at 1, 3, 5, 10 and 15 years using the log-rank test. Results: Mean age was 66.9 ± 13 years and mean follow-up was 5.3 ± 4.7 years. A total of 48.9% had LR. In-hospital mortality was 10% (LR: 6% vs. ER: 13.8%, p < 0.01). ER, NYHA class, salvage surgery and additional procedures were predictors of increased mortality in unmatched data. Propensity-matched data showed no difference in TIA/stroke rates, LOS, inpatient mortality or composite outcomes. LR had better survival (LR: 77.1% vs. ER: 51.4%, p < 0.001). ER (OR: 1.97, 95% CI: 1.27, 3.08, p = 0.003) was a significant predictor of worse long-term survival. At 15 years, aortic re-operation was 17% and freedom from re-operation and death was 42%. Conclusions: Type A aortic dissection repair has high mortality and morbidity, although results have improved over two decades. ER was a predictor of worse perioperative results and long-term survival.

The action of phytochemicals in biofilm control.

Covering: 2009 to 2021Antimicrobial resistance is now rising to dangerously high levels in all parts of the world, threatening the treatment of an ever-increasing range of infectious diseases. This has becoming a serious public health problem, especially due to the emergence of multidrug-resistance among clinically important bacterial species and their ability to form biofilms. In addition, current anti-infective therapies have low efficacy in the treatment of biofilm-related infections, leading to recurrence, chronicity, and increased morbidity and mortality. Therefore, it is necessary to search for innovative strategies/antibacterial agents capable of overcoming the limitations of conventional antibiotics. Natural compounds, in particular those obtained from plants, have been exhibiting promising properties in this field. Plant secondary metabolites (phytochemicals) can act as antibiofilm agents through different mechanisms of action from the available antibiotics (inhibition of quorum-sensing, motility, adhesion, and reactive oxygen species production, among others). The combination of different phytochemicals and antibiotics have revealed synergistic or additive effects in biofilm control. This review aims to bring together the most relevant reports on the antibiofilm properties of phytochemicals, as well as insights into their structure and mechanistic action against bacterial pathogens, spanning December 2008 to December 2021.

Outcomes of Heller Myotomy for Esophageal Achalasia: Lessons From a 48-Year Prospective Experience With 4 Different Techniques.

OBJECTIVE: To provide information on long-term outcomes of Heller myotomy for esophageal achalasia with or without an antireflux fundoplication. BACKGROUND: Since the adoption of the Heller myotomy, surgeons have modified the original technique in order to balance the cure of dysphagia and the consequent cardial incontinence. METHODS: Totally, 470 patients underwent primary Heller myotomy between 1955 and 2020. A long abdominal myotomy (AM) was performed in 83 patients, the Ellis limited transthoracic myotomy (TM) in 30, the laparotomic Heller-Dor (L-HD) in 202, the videolaparoscopic Heller-Dor (VL-HD) in 155. The HD was performed under intraoperative manometric assessment. Starting on 1973 these patients underwent a prospective follow-up program of timed lifelong clinical, radiological, endoscopic evaluations. RESULTS: Median follow-up time was 23.06 years [interquantile range (IQR): 15.04-32.06] for AM, 29.22 years (IQR: 13.46-40.17) for TM, 14.85 years (IQR: 11.05-21.56) for L-HD and 7.51 years (IQR: 3.25-9.60) for VL-HD. In AM, relapse of dysphagia occurred in 25/71 (35.21%), in TM in 11/30 (36.66%), in LH-D in 10/201 (4.97%), in VL-HD in 3/155 (1.93%). Erosive-ulcerative esophagitis was diagnosed for AM in 28.16%, for TM in 30%, for L-HD in 8.45%, for VL-HD in 2.58%. Overall, the outcome was satisfactory in 52.11% for AM, 41.9% for TM, 89.05% for L-HD, 96.12% for VL-HD. CONCLUSIONS: The Dor fundoplication drastically reduces postmyotomy gastroesophageal reflux. The Heller-Dor operation is a competitive option for the cure of esophageal achalasia if this operation is performed according to the rules of surgical physiology learned by means of intraoperative manometry.